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Padmaa et al. European Journal of Biomedical and Pharmaceutical Sciences
www.ejbps.com
349
IN VITRO AND IN SILICO ANTICANCER ACTIVITY OF NEGUNDOSIDE ISOLATED
FROM LEAVES OF VITEX NEGUNDO LINN
Padmaa M. Paarakh*1, Dileep Chandra Sreeram2, Shruthi S.D.3 and Sujan Ganapathy P.S.4
1Dept of Pharmacognosy, The Oxford College of Pharmacy, Hongasandra, Bangalore, Karnataka.
2R & D], Himalaya Wellness Company, Bangalore.
3Microbiology and Cell Biology Department, Indian Institute of Science, Bangalore, Karnataka.
4Research and Development Centre, Olive Life Sciences Pvt. Ltd., Nelamangala, Bangalore 562123, Karnataka, India.
Article Received on 22/12/2016 Article Revised on 12/01/2017 Article Accepted on 01/02/2017
INTRODUCTION
Cancer is one of the highest impacting diseases
worldwide with significant morbidity and mortality
rates. The current known therapies are based on radio
and chemotherapies and although in many cases, the
patients have their health re-established, the treatment
is very painful since their immunological system is
severely compromised, because these procedures are
not cells selective.[1] Substantial advances have been
made in understanding the key roles of receptor
tyrosine kinase (RTK) in the signalling pathways that
govern fundamental cellular processes, such as
proliferation, migration, metabolism, differentiation
and survival. In the normal cells RTK activity is tightly
controlled.When they are mutated or structurally
altered, they become potent oncoproteins which leads
to abnormal activation of RTKs in transformed cells
has been shown to be causally involved in the
development and progression of many human
cancers.[2] The cost of treatment is very high and with
lot of side effects. In order to find new natural sources
that are biologically active substances from plants have
acquired immense attention. A number of studies have
been carried out on various plants, vegetables and
fruits because they are rich sources of
phytoconstituents which prevent free radical damage
thereby reducing risk of chronic diseases viz., cancer,
cardiovascular diseases etc. This beneficial role of
plants has led to increase in the search for newer plant
based sources for the treatment of diseases like cancer.
One such plant is Vitex negundo Linn.
Vitex negundo Linn., commonly known as Five-leaved
Chaste tree or Monk’s Pepper (Hindi —Sambhalu,
Nirgundi) is used as medicine fairly throughout the
greater part of India and found mostly at warmer zones
and ascending to an altitude of 1500m in outer Western
Himalayas.[3-4] In traditional system of medicine, the
plant is used as bitter, acrid, astringent, cephalic,
stomachic, antiseptic, alterant, thermogenic,
depurative, rejuvenating, ophthalmic, anti-gonorrhoeic,
antiinflammatory, antipyretic and useful in bronchitis,
asthma and enlargement of spleen. Leaves are
aromatic, bitter, acrid, astringent, anodyne, anti-
inflammatory, antipyretic or febrifuge, tranquillizer,
bronchial smooth muscle relaxant, anti-arthritic,
antihelmintic and vermifuge.[3] It is reported to
possess analgesic, anti-inflammatory, antioxidant,
anticonvulsant, antimalarial, antifiliarial,
SJIF Impact Factor 4.382
Research Article
ejbps, 2017, Volume 4, Issue 02, 349-354.
European Journal of Biomedical
AND Pharmaceutical sciences
http://www.ejbps.com
ISSN 2349-8870
Volume: 4
Issue: 2
349-354
Year: 2017
*Corresponding Author; Dr. Padmaa M. Paarakh
Dept of Pharmacognosy, The Oxford College of Pharmacy, Hongasandra, Bangalore, Karnataka.
ABSTRACT
Vitex negundo Linn.[Verbenaceae], commonly known as Five-leaved Chaste tree or Monk’s Pepper (Hindi —
Sambhalu, Nirgundi) is used as medicine fairly throughout the greater part of India. The aim of present study is to
isolate negundoside and evaluate anticancer activity by in vitro and in silico method. Negundoside was isolated by
column chromatography from ethyl acetate fractionation of methanol extract of leaves of V.negundo. Negundoside
was characterized by UV,IR, 1H-NMR, 13C-NMR and Mass spectrum. Standardization of negundoside was done
by HPTLC fingerprinting. In vitro anticancer activity was done using HeLa cell lines by MTT assay at different
concentrations ranging from 20-100 µg/ml and in silico docking studies using enzyme EGFR tyrosine kinase.
Fingerprinting of isolated negundoside were done by HPTLC method. The IC50 value was found to be 62.69 µg/ml
in in vitro anticancer activity in HeLa Cell lines. Negundoside was subjected to molecular docking studies for the
inhibition of the enzyme EGFR tyrosine kinase, which is one of the targets for inhibition of cancer cells. It has
shown -7.32 kJ mol-1 binding and -11.32 kJ mol-1 docking energy with five hydrogen bonds. Negundoside has
shown to possess anticancer activity both in vitro and in silico studies.
KEYWORDS: In vitro anticancer activity; In silico docking studies; Isolation; Negundoside; Vitex negundo.
Padmaa et al. European Journal of Biomedical and Pharmaceutical Sciences
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350
hepatoprotective, antibacterial and antifertility
activities.[5]
The aim of the present study is to isolate negundoside
from dried leaves of Vitex negundo and perform in
vitro MTT assay and in silico activity to prove its
anticancer activity.
MATERIAL AND METHODS
Plant Material
The dried leaves of Vitex negundo (Verbenaceae) were
collected, identified and authenticated by Dr
Shiddamallayya N (SMPU/NADRI/BNG/345) at
National Ayurveda Dietetics Research Institute,
Bengaluru, Karnataka. A voucher specimen was
deposited in the Herbarium of Department of
Pharmacognosy, The Oxford College of Pharmacy,
Bangalore. The leaves were dried under normal
environmental conditions. The dried leaves were
powdered and stored in a closed container for further
use.
Drugs and Chemicals
DMEM medium (GIBCO), heat-inactivated fetal
bovine serum (FBS), trypsin, ethylene-
diaminetetraacetic acid (EDTA),PBS and MTT were
purchased from Hi media and Sigma Chemicals. All
chemicals and reagents used in this study were at least
of analytical grade.
Extraction and Isolation Procedure
The dried leaves of V. negundo (350 g) was refluxed
with methanol (1.5 l) for 1 h. Filter and repeat the
process of reflux by adding methanol(1.5 l) to the
marc. Distill the combined methanol extract to remove
the solvent and dry the concentrated residue under
vacuum to get a thick green paste(35 g). Dissolve the
extractive in 1litre of water and carry out partitioning
(liquid-liquid) with ethyl acetate and water (1:1)three
times. Concentrate the ethyl acetate layer under
vacuum. Ethyl acetate fraction was subjected to
column chromatography over silica gel (60-120 mesh)
using n-hexane with increasing percentage of
chloroform and methanol. The fractions eluted with
20-30 % methanol in chloroform were collected and
concentrated under vacuum to get a residue (1 g)
which was further chromatographed on a polystyrene
gel (Diaion HP-20) (60 g) column. Elute the column
by gradient elution using water with increasing
percentage of acetone. The fractions eluted with 10 to
15 % acetone in water were combined and concentrate
under vacuum to get enriched fraction of negundoside
(0.2g). Crystallize the fraction in methanol to get
negundoside (100mg).
Charcterization of Negundoside
The structure of Negunoside was characterized by UV,
IR, NMR, Mass spectrum. HPTLC fingerprinting was
done to confirm the presence and purity of
negundoside.
Chromatographic Finger Printing of the Dried Leaf
of V. negundo using Negundoside
TLC Identity Test
Weigh 2 g of coarsely powdered drug and transfer to a
250-ml conical flask. Extract with 50 ml of methanol
by refluxing for about 20 min and filter. Repeat the
process 4 to 5 times till the raw material is completely
exhausted or till the extract is colourless. Combine the
extracts and concentrate to a volume of about 100 ml,
cool to room temperature. Use the solution for TLC
profiling. Standard solution was prepared by dissolving
5 mg of negundoside in 10 ml of methanol. Solvent
system used was Ethyl acetate : Methanol : Water :
Acetic acid (7.8:1.2:0.7:0.3). Apply 20 µl of test
solution and 5 µl of standard solution separately on a
precoated silica gel 60F254TLC plate (E. Merck) of
uniform thickness (0.2 mm). Develop the plate in the
solvent system till the solvent rises to a distance of 8
cm. Visualization was done after spraying with
anisaldehyde-sulphuric acid reagent and followed by
heating at 105o C for 5 to 10 min. The Rf value and
colour of the resolved bands were noted.
In vitro anticancer activity using HeLA cell lines by
MTT assay
Cell culture
HeLa cell line was maintained in DMEM medium
(GIBCO) supplemented with 10% (v/v) heat-
inactivated fetal bovine serum (FBS) and 1% antibiotic
solution (penicillin 100Uml−1 and streptomycin
100µgml−1) at 37◦C in a humidified atmosphere of 95%
air/5% CO2. The medium was changed every second
day, and cells were subcultured when confluency reach
to 95% by 0.25% trypsin containing 0.02% ethylene-
diaminetetraacetic acid (EDTA) in PBS for 3 min at
37◦C.
MTT Assay
The MTT assay was carried out as described
previously to measure cell viability6. Ten thousand
cells in 100μL of DMEM media were seeded in the
wells of a 96-well plate. After 24 h, existing media was
removed and 100 μL of various concentrations of
compound was added and incubated for 48 h at 37 °C
in a CO2 incubator. Control cells were supplemented
with 0.05% DMSO vehicle. At the 48th hour of
incubation, MTT (3-(4,5-dimethylthaizol-2-yl)-2,5-
diphenyltetrazolium bromide- supplied from Sigma,
10μL of 5 mg/mL) was added to the plate. The
contents of the plate were pipetted out carefully, the
formazan crystals formed were dissolved in 100 μL of
DMSO, and the absorbance was measured at 550 nm
in a microplate reader (Tecan, infinite F200 Pro).
Experiments were performed in triplicate, and the
results were expressed as mean of percentage
inhibition. A graph of the concentration versus
percentage growth inhibition was plotted, and the
concentration at which 50% cell death occurred was
considered as the IC50 value. Before adding MTT,
bright field images (Olympus 1X81, cellSens
Padmaa et al. European Journal of Biomedical and Pharmaceutical Sciences
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351
Dimension software) were taken for visualizing the
cell death.
In silico activity:Molecular Docking studies
The three dimensional structure of target protein EGFR
tyrosine kinase(PDB ID:2J5F) was downloaded from
PDB (www.rcsb.org/pdb) structural database. This file
was then opened in SPDB viewer edited by removing
the heteroatoms, adding C terminal oxygen. The active
pockets on target protein molecule were found out
using CASTp server7. The ligands were drawn using
ChemDraw Ultra 6.0 and assigned with proper 2D
orientation (ChemOffice package). 3D coordinates
were prepared using PRODRG server.[8] Autodock
V3.0 was used to perform Automated Molecular
Docking in AMD Athlon (TM)2x2 215 at 2.70 GHz,
with 1.75 GB of RAM. AutoDock 3.0 was compiled
and run under Microsoft Windows XP service pack 3.
For docking, grid map is required in AutoDock, the
size of the grid box was set at 102, 126 and 118 Å (R,
G, and B), and grid center -58.865, -8.115, -24.556 for
x, y, and z-coordinates. All torsions were allowed to
rotate during docking. The Lamarckian genetic
algorithm and the pseudo-Solis and Wets methods
were applied for minimization, using default
parameters.[9]
RESULTS
The Characterization of the Negundoside[Figure
No.1]
Physical data: Negundoside is an amorphous white
powder, mp 156o, lit2 mp 155-159°;
[α] -128.24o, lit2[α] -119.2o;Soluble in chloroform
and methanol; insoluble in water.
Molecular formula: C23H28O12; Molecular weight:
496;Elemental composition: C 55.64%; H 5.68%; O
38.67%.
Spectral Data
UV-VIS spectrum shows absorption at 254 nm(ε = 1.8
x 104). The UV-VIS spectrum indicates the presence of
chromophoric system in the molecule.The IR spectrum
shows a broad band at 3309 cm-1 due to presence of
hydroxyl group. A band at 1698 cm-1 indicates the
carbonyl group in the molecule. The band at 1514 cm-1
is due to aromaticity in the molecule and band at 1273
cm-1 can be assigned to a stretching at C–O–C. 1H-
NMR spectrum (DMSO-d6-300 MHz): The signal at δ
1.13 (3H, s,C8H), 1.29 (1H, m,C5aH), 1.55 (2H,
m,C6H), 1.99 (1H, dd,C7aH, J=9.6, 3.3 Hz), 2.04 (1H,
m,C5bH), 2.73 (1H, m,C4aH), 3.52 (1H, m,C6′aH), 3.76
(1H, dd,C6′bH, J=11.1, 1.5 Hz), 4.74 (1H, t,C2′H,
J=9.0Hz), 4.85 (1H, d,C1′H, J=8.1Hz), 5.30 (1H,
d,C1H, J=3.0Hz), 6.81 (2H, d,C3′′H, C5′′H, J=8.7Hz),
7.04 (1H, s,C3H), 7.73 (2H, d,C2′′H, C6′′H, J=8.4Hz).
The 1H-NMR spectrum shows singlet at δ 1.13 due to
methyl protons at C8. Multiplets at δ 1.29 and 1.55
correspond to protons at C5a and C6, respectively.
Proton at C7a appears as doublet of doublet at δ 1.99.
Protons at C5b, C4a and C6′a were observed as multiplets
at δ2.04, 2.73 and 3.52, respectively. Protons at C6′b
and C2′are observed as doublet of doublet and triplet at
δ 3.76 and 4.74, respectively.Protons at C1′ and C1
appear as doublets at δ4.85 and 5.30, respectively.
Doublet at δ 6.81corresponds to protons at C3′′ and C5′′.
Olefinic proton at C3 is observed as singlet at δ 7.04.
The protons at C2′′ and C6′′ appear as doublet at δ7.73.
13C NMR spectrum (CD3OD -300 MHz): the signals at
22.97, 28.79, 29.82, 39.87, 51.03, 61.38, 70.41,
73.57, 74.69, 77.12, 78.48, 93.71, 96.51, 112.40,
114.76, 120.86, 131.49, 149.76, 161.90, 165.93,
168.60. The assignment of 13C-NMR is given in the
Table No.1.
The HPTLC fingerprinting confirmed the presence of
negundoside [Table No.2, 3 and Figure No. 2,3]. A
grey and reddish-pink coloured bands are observed at
(Rf 0.57) corresponding to negundoside is visible in
both the test and standard solution tracks under UV at
254 nm and after derivatization.
In vitro anticancer activity on HeLa Cell Lines
The MTT values obtained demonstrated that
Negundoside has good cytotoxic effect. The IC50 value
was found to be 62.69 µg/ml. Microscopy images
representing the cell death caused by the compounds
are as seen in Figure No.4. It is very clear that it is very
good cytotoxic agent.
In silico molecular docking studies
The tyrosine kinase receptors have multidomain
extracellular Ligands for specific Ligand, a signal pass
transmembrane hydrophobic helix and tyrosine kinase
domain. The receptor tyrosine kinases are not only cell
surfaces transmembrane receptors, but are also
enzymes having kinase activity.[10] In cancer,
angiogenesis is an important step in which new
capillaries develop and develop for supplying a
vasculature to provide nutrient and removing waste
material. So tyrosine kinase inhibitor as an anti-
angiogenic agent is new cancer 4 therapy. Developing
natural drugs and prodrugs as inhibitor is today’s trend.
Low molecular weight substances, which inhibit
tyrosine kinase phosphorylation block signaling
pathway, initiated in the extracellular part of
receptor11.Since, the type I receptor tyrosine kinase is a
major regulator of several distinct and diverse cellular
pathways we have evaluated it as a target.
Negundoside was taken and docked to get the best
conformer. Results were compared for the binding
energy, docking energy and number of hydrogen bonds
formed. According to the docking results (Table No.4),
it has binding energy of: -7.32, with five hydrogen
bonds formed.
Padmaa et al. European Journal of Biomedical and Pharmaceutical Sciences
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352
Molecular docking with EGFR tyrosine kinase domain
revealed that, our compound have inhibitory capability
and thereby exhibiting interactions with one or the
other amino acids in the active pockets as shown in
Figure No.5. The topology of the active site of EGFR
tyrosine kinase was similar in all synthesized
molecules, which is lined by interacting amino acids as
predicted from the ligplot (Figure No.5).In in vitro
studies the molecule emerged as more active against
the cell line used.
Table No.2: HPTLC Details of Test Solution of Vitex negundo Dried Leaf at 254 nm.
Rf value
Colour of the band
0.52
Grey
0.57
Grey(Negundoside)
0.94
Grey
Table No.3: HPTLC Details of Test Solution of Vitex negundo dried Leaf after Derivatization.
Rf value
Colour of the band
0.37
Light pink
0.52
Reddish-brown
0.57
Reddish-pink (Negundoside)
Table No.4: Molecular docking results of Negundoside with EGFR tyrosine kinase.
Molecule
Binding
energy
Docking
energy
Inhibitory
constant
Intermol
energy
H-bonds
Bonding
NGD
-7.32
-11.32
4.3e-006
-9.81
5
NGD::DRG:OAG:TK:A:ARG831:HH12
NGD::DRG:HBD:TK:A:ASP770:OD2
NGD::DRG:OBI:TK:A:ARG776:HE
NGD::DRG:H22:TK:A:SER768:O
NGD::DRG:HAR:TK:A:VAL769:O
Table No.1: 13C NMR Assignments of negundoside.
No
DEPT
(ppm)
Assignment
1.
CH3
22.97
C8
2.
CH2
28.79
C5
3.
CH
29.82
C4a
4.
CH2
39.87
C6
5.
CH
51.03
C7a
6.
CH2
61.38
C6′
7.
CH
70.41
C4′
8.
CH
73.57
C3′
9.
CH
74.69
C2′
10.
CH
77.12
C5′
11.
Quaternary
78.48
C7
12.
CH
93.71
C1
13.
CH
96.51
C1′
14.
Quaternary
112.40
C4
15.
CH
114.76
C3′′, C5′′
16.
Quaternary
120.86
C1′′
17.
CH
131.49
C2′′, C6′′
18.
CH
149.76
C3
19.
Quaternary
161.90
C4′′
20.
Quaternary
165.93
C7′′
21.
Quaternary
168.60
C9
Padmaa et al. European Journal of Biomedical and Pharmaceutical Sciences
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353
Figure No.1:Strucuture of the compound
Negundoside.
1 2 3 4
Figure No.2: HPTLC profile of test solution of Vitex
negundo dried leaf at 254 nm.
1 : Negundoside standard; 2-4 : Test solution
1 2 3 4
Figure No.3: HPTLC profile of test solution of Vitex
negundo dried leaf after derviatization.
1: Negundoside standard; 2-4: Test solution
Figure No.4: Anticancer activity of Negundoside
showing cell death; A-control; B-treated
Figure No.5:3D structure of EGFR tyrosine kinase from
PDB (A); Interacting amino acids as predicted from the
ligplot (B); Enfolding of Negundoside in the active
pocket (C).
CONCLUSION
Negunoside has shown to possess anticancer activity
both in vitro and in silico studies. The IC50 value was
found to be 62.69 µg/ml and in silico studies, it has more
number of hydrogen bonds with minimum binding and
docking energy and may be considered as inhibitor of
EGFR tyrosine kinase. More experiments are required to
understand the exact mechanism by which the cells are
affected. It is important to correlate the structure of these
compounds with their biological effect, which will be
valuable to propose new lead compounds with better
cytotoxic potential.
ACKNOWLEDGEMENT
The authors are grateful to Department of
Pharmacognosy, The Oxford College of Pharmacy,
Bangalore, for providing the facilities for carrying out
the entire experiment.
Ethical Issues
There is none to be applied.
Conflict of Interest
None to be declared.
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